DESCRIPTION (Applicant's abstract): Mechanical ventilation (MV) is used clinically to sustain ventilation in patients who are incapable of independently maintaining adequate alveolar ventilation. Unfortunately, the withdrawal of MV, or weaning, can be difficult in a large number of cases. Strong evidence exists that MV-induced respiratory muscle weakness contributes significantly to these difficulties in weaning. Indeed, we have recently demonstrated that prolonged MV results in diaphragmatic atrophy and a significant reduction in diaphragmatic maximal force production. Further, we have observed that prolonged MV results in oxidative injury (i.e. protein oxidation) to the diaphragm; this is significant because oxidized proteins become targets for proteases. The mechanisms responsible for this MV-induced atrophy and protein oxidation are unknown and comprise the focus of our proposed experiments. To determine the factors that contribute to diaphragmatic atrophy during prolonged MV, we will test the following hypotheses: 1a) MV-induced diaphragmatic atrophy occurs due to a decrease in synthesis of muscle proteins as well as an increased rate of proteolysis; 1b) proteolysis is the major contributor to diaphragmatic protein loss during prolonged MV; 2a) The increased activity of calpain, lysosomal, and ATP ubiquitin-dependent proteases are collectively responsible for the protein degradation observed in diaphragms from MV animals; and 2b) Although calpain, lysosomal, and ATP-ubiquitin-dependent proteases all contribute to diaphragmatic protein loss during MV, the ATP-ubiquitin-dependent and calpain proteolytic pathways are dominant. To resolve which chemical pathways are responsible for diaphragmatic protein oxidation during MV we will test the hypothesis that MV-induced protein oxidation in the diaphragm is caused by several reactive chemical species including hypochlorous acid, tyrosyl radicals and hydroxyl radicals. To test these postulates, we will perform both in vitro and in vivo studies using an animal model and utilize the tools of molecular biology, biochemistry, and physiology. These experiments will improve our understanding of the mechanisms associated with MV-induced diaphragmatic atrophy. The long-term goal of our experiments is to provide the knowledge required to develop clinical strategies to oppose the deleterious effects of MV on respiratory muscles.